LTB4 as vaccine adjuvant

ABSTRACT

The present invention relates to a vaccine adjuvant for enhancing immune response of an individual to a vaccine, which comprises an immune-enhancing effective amount of an LTB4 agent in association with a pharmaceutically effective vaccine carrier.

BACKGROUND OF THE INVENTION

(a) Field of the Invention

The invention relates to the use of a leukotriene B4 (LTB₄) agent as anadjuvant to vaccine preparations.

(b) Description of Prior Art

Vaccines have been used for decades for the prevention of diseases inhumans. The efficacy of any vaccine preparation largely depends on itsimmunogenicity, i.e., their ability to induce strong humoral andcellular immunity. However, many vaccines currently in use have moderateefficacy due to their weak immunogenicity. Because of this, severalattempts have been made to supplement vaccine preparations withadjuvants in order to increase the ability of a given vaccine to inducea strong immunity. Unfortunately, most of the substance used asadjuvants have undesirable side effects, which prevent their use inhumans.

Adjuvants have been used extensively to improve the generation of animmune response following immunization with a particular antigen,especially in laboratory animals. However, classical and effectiveadjuvants, such as the Freund's adjuvant, cause undesirable sideeffects, which prevent their use in humans. Less toxic adjuvants, suchas aluminum hydroxide (alum), although relatively well tolerated, do notoffer the same degree of immunopotentiation as the Freund's adjuvant.

Thus, because of a lack of effective adjuvant, vaccines for human useare often poorly immunogenic and multiple immunization regimens arerequired to achieve proper protection against a given pathogen. Inaddition, long-lasting immunity is often lost in absence of repeatedimmunization. Intense efforts are therefore devoted to theidentification of new effective adjuvants to complement currently usedvaccines.

Leukotriene B4 (LTB₄) is a known natural molecule. LTB₄ is a metaboliteof arachidonic acid derived from the 5-lipoxygenase pathway. LTB₄ hasmany reported biological properties. In particular, LTB₄ is consideredas a potent pro-inflammatory compound; its most important biologicalactivity is its chemotactic and chemokinetic effects on leukocytes.Indeed, LTB₄ has been shown to be a potent chemoattractant for humanpolymorphonuclear leukocytes, monocytes and macrophages, both in vitroand in vivo. LTB₄ also activates other leukocyte functions such asdegranulation and superoxide anion synthesis. Because of thesepro-inflammatory effects, LTB₄ is considered as a putative component indefense mechanisms. Moreover, LTB₄ is synthesized by inflammatory cellssuch as polymorphonuclear leukocytes, monocytes and macrophages and isalso synthetized by B lymphocytes.

The effects of LTB₄ on B cell activity have been previously studiedunder in vitro experimental conditions. B lymphocyte proliferation,expression of activation markers such as CD23 and immunoglobulinsecretion were evaluated. Most studies reached similar conclusionsstating that LTB₄ by itself, in absence of exogenous cytokines orstimuli (Protein A or S. aureus), had no effects on B cell proliferationor immunoflobulin synthesis (IgG or IBM) (Yamaoka, K. A., et al., 1989,J. Immunol. 143: 1996-2000; Dugas, B., et al., 1990, J. Immunol.145:3406-3411; Odlander, B., et al., 1989, Int. J. Tiss. Reac.XI(6):277-289). The only observable effects of LTB₄ on B lymphocyteswere recorded when combined to a stimulating agents (Protein A or S.aureus) and cytokines. A conflicting paper also reports that LTB₄ had aninhibitory effects on the synthesis of immunoglobulin from B lymphocytes(Rola-Plecszczynski, M., et al., 1982, Biochem. Biophys. Res. Commun.108:1531-1537). It thus appear that LTB₄ by itself does not stimulate Bcell functions and perhaps may even negatively influence it underdefined experimental conditions.

It would be highly desirable to be provided with an adjuvant withgreater efficacy than the currently used hydroxide aluminum basedadjuvant and which would not present the undesirable side effects of themore potent Freund's adjuvant.

SUMMARY OF THE INVENTION

The present invention provides a mean of enhancing immune response,particularly humoral immune response, by concomitant administration ofan immune-enhancing effective amount of a LTB₄ agent to vaccinepreparations.

One aim of the present invention is to provide an adjuvant and usethereof to current and future vaccine preparations allowing them to bemore effective in generating protective immunity against pathogens.

Another aim is to provide an adjuvant to vaccine preparations destinedto immunosuppressed individuals.

In accordance with the present invention there is provided the use of anLTB₄ agent as an adjuvant, for example, with vaccines against the Flu(Influenza) and Tuberculosis (BCG), among others.

In accordance with the present invention there is provided a vaccineadjuvant for enhancing immune response of an individual to a vaccine,which comprises an immune-enhancing effective amount of an LTB₄ agent inassociation with a pharmaceutically effective vaccine carrier.

In accordance with the present invention there is provided a vaccinepreparation causing enhanced immune response from an individual, whichcomprises an immune-enhancing effective amount of an LTB₄ agent inassociation with a vaccine preparation.

In accordance with the present invention there is provided the use of animmune-enhancing effective amount of an LTB₄ agent for the preparationof a vaccine causing enhanced immune response from an individual, whichcomprises an immune-enhancing effective amount of an LTB₄ agent inassociation with a vaccine preparation.

More preferably, the immune response, enhanced by the vaccine of thepresent invention, is a humoral immune response.

More preferably, the vaccine of the present invention isimmunoprotective against a pathogen selected from the group consistingof Influenza and Tuberculosis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the effects of concomitant administration of LTB₄ tothe Fluviral vaccine on anti-Influenza antibody generation in BALB/cmice.

FIG. 2 illustrates the effects of concomitant admistration of LTB₄ tothe BCG vaccine on anti-mycobacterium tuberculosis antibody generationin BALB/c mice.

FIG. 3 illustrates the effects of prolonged LTB₄ administration onanti-cytomegalovirus (CMV) antibody generation during acute CMVinfection in BALB/c mice.

DETAILED DESCRIPTION OF THE INVENTION i) LTB₄

The term “leukotriene B4 (LTB₄) agent” in accordance with the presentinvention is intended to mean LTB₄ or certain structurally relatedpolyunsaturated fatty acids, or substances structurally unrelated tofatty acids, which stimulate the synthesis of LTB₄ or other LTB₄ agentsby cells, or mimic their biological activity. They are either naturalsubstances or analogs of such natural substances. All of the LTB₄ agentscan be obtained by chemical synthesis by methods described in theliterature and most are commercially available.

As used herein, the term “LTB₄ agent” is intended to mean one or more ofthe following polyunsaturated fatty acids, which in addition to LTB₄itself, are analogs of LTB₄, or precursors or metabolites of LTB₄ orLTB₄ analogs: LTB₄, 14,15-dihydro-LTB₄, 17,18-dehydro-LTB₄,19-hydroxy-LTB₄, 20-hydroxy-LTB₄ and their 5(R)-hydroxy, 5-keto,5(S)hydroperoxy, 5(R)-hydroperoxy and 5-deoxy analogs; LTA₄;14,15-dihydro-LTA₄, 17,18-dehydro-LTA₄;5(S)-hydroxy-6,8,11,14(E,Z,Z,Z)-eicosatetraenoic acid (“5-HETE”),14,15-dihydro-5-HETE, 17,18-dehydro-5-HETE, and their 5(R)-hydroxy,5-keto, 5(S)-hydroperoxy, 5(R)-hydroperoxy analogs;12(R)-hydroxy-5,8,10,14(Z,Z,E,Z)-eicosatetraenoic acid (“12-HETE”),5,6-dihydro-12-HETE, 14,15-dihydro-12-HETE, 17,18-dehydro-12-HETE andtheir 12(S)-hydroxy, 12-keto, 12(S)-hydroperoxy and 12(R)-hydroperoxyanalogs and 12-oxo-5,8,10(Z,Z,E)-dodecatrienoic acid,15(S)-hydroxy-5,8,11,13(Z,Z,Z,E)-eicosatetraenoic acid (“15-HETE”),5,6-dihydro-15-HETE, 17,18-dehydro-15-HETE and their 15(R)-hydroxy,15-keto, 15(S)-hydroperoxy, and 15(R)-hydroperoxy analogs.

The term LTB₄ agent also includes other derivatives of polyunsaturatedfatty acids; some are derived from the cyclooxygenase pathways, thelipoxygenase pathways (5-, 12- and 15-lipoxygenases) or the cytochromeP450 pathways; others are isomers, analogs or derivatives of naturallyformed compounds: 12(S)-hydroxy-5,8,10(Z,E,E)-heptadecatrienoic acid;leukotrienes C₄, D₄ and E₄ and their 14,15-dihydro or 17,18-dehydroanalogs; N-acyl or N-alkyl derivatives of leukotrienes C₄, D₄ and E₄,and their 14,15-dihydro or 17,18-dehydro analogs; all isomeric5,12-dihydroxy-6,8,10,14-eicosatetraenoic acids and their 14,15-dihydroor 17,18-dehydro analogs; all isomeric5,6-dihydroxy-7,9,11,14-eicosatetraenoic acids and their 14,15-dihydroor 17,18-dehydro analogs; all isomeric5,15-dihydroxy-6,8,11,13-eicosatetraenoic acids (including5(S),15(S)-dihydroxy-6,8,11,13(E,Z,Z,E)-eicosatetraenoic acid) and their17,18-dehydro analogs; all isomeric8-hydroxy-11(12)-epoxy-5,9,14-eicosatrienoic acids (including hepoxilinA₃) and their 5,6-dihydro or 14,15-dihydro or 17,18-dehydro analogs; allisomeric 10-hydroxy-11(12)-epoxy-5,8,14-eicosatrienoic acids (includinghepoxilin B₃) and their 5,6-dihydro or 14,15-dihydro or 17,18-dehydroanalogs; all isomeric 8,11,12-trihydroxy-5,9,14-eicosatrienoic acids(including trioxilin A₃) and their 5,6-dihydro or 14,15-dihydro or17,18-dehydro analogs; all isomeric10,11,12-trihydroxy-5,8,14-eicosatrienoic acids (including trioxilin B₃)and their 5,6-dihydro or 14,15-dihydro or 17,18-dehydro analogs; allisomeric 11(12)-epoxy-5,7,9,14-eicosatetraenoic acids and their14,15-dihydro or 17,18-dehydro analogs; all isomeric11,12-dihydroxy-5,7,9,14-eicosatetraenoic acids and their 14,15-dihydroor 17,18-dehydro analogs; all isomeric8(9)-epoxy-5,10,12,14-eicosatetraenoic acids and their 5,6-dihydro or17,18-dehydro analogs; all isomeric8,9-dihydroxy-5,10,12,14-eicosatetraenoic acids and their 5,6-dihydro or17,18-dehydro analogs; all isomeric8,15-dihydroxy-5,9,11,13-eicosatetraenoic acids and their 5,6-dihydro or17,18-dehydro analogs; all isomeric14(15)-epoxy-5,8,10,12-eicosatetraenoic acids and their 5,6-dihydro or17,18-dehydro analogs; all isomeric14,15-dihydroxy-5,8,10,12-eicosatetraenoic acids and their 5,6-dihydroor 17,18-dehydro analogs; all isomeric5-hydroxy-14(15)-epoxy-6,8,10,12-eicosatetraenoic acids and their17,18-dehydro analogs; all isomeric5,14,15-trihydroxy-6,8,10,12-eicosatetraenoic acids (including lipoxinB₄) and their 17,18-dehydro analogs; all isomeric5,6,15-trihydroxy-7,9,11,13-eicosatetraenoic acids (including lipoxinA₄) and their 17,18-dehydro analogs; all isomeric5(6)-epoxy-15-hydroxy-7,9,11,13-eicosatetraenoic acids and their17,18-dehydro analogs; all isomeric 5-hydroxy-6,8,11,14-eicosatetraenoicacids and their 14,15-dihydro or 17,18-dehydro analogs; all isomeric8-hydroxy-5,9,11,14-eicosatetraenoic acids and their 5,6-dihydro or14,15-dihydro or 17,18-dehydro analogs; all isomeric9-hydroxy-5,7,11,14-eicosatetraenoic acids and their 14,15-dihydro or17,18-dehydro analogs; all isomeric11-hydroxy-5,8,12,14-eicosatetraenoic acids and their 5,6-dihydro or17,18-dehydro analogs; all isomeric12-hydroxy-5,8,10,14-eicosatetraenoic acids and their 5,6-dihydro or14,15-dihydro or 17,18-dehydro analogs; all isomeric15-hydroxy-5,8,11,13-eicosatetraenoic acid and their 5,6-dihydro or17,18-dehydro analogs; all isomeric 9-hydroxy-10,12-octadecadienoicacids; all isomeric 13-hydroxy-9,11-octadecadienoic acids;12(R)-hydroxy-5,8,14(Z,Z,Z)-eicosatrienoic acid; all isomeric 5(6)oxido-or 5,6-dihydroxy-8,11,14-eicosatrienoic acids and their 14,15-dihydro or17,18-dehydro analogs; all isomeric 8(9)-oxido- or8,9-dihydroxy-5,11,14-eicosatrienoic acids and their 5,6-dihydro or14,15-dihydro or 17,18-dehydro analogs; all isomeric 11(12)-oxido- or11,12-dihydroxy-5,8,14-eicosatrienoic acids and their 5,6-dihydro or14,15-dihydro or 17,18-dehydro analogs; all isomeric 14(15)-oxido- or14,15-dihydroxy-5,8,11-eicosatrienoic acids and their 5,6-dihydro or17,18-dehydro analogs.

The term LTB₄ also includes variants which are non-covalently modifiedfatty acids such as the sodium or the potassium salts of the LTB₄agents.

The term LTB₄ agent also includes variants where a modification isintroduced into the molecule by reacting targeted functional groups ofthe fatty acid with an organic derivatizing agent that is capable ofreacting with the selected functional group (yielding for example, esterand ether derivatives of LTB₄ agent) or to cause intramolecularrearrangement (such as the formation of lactones with hydroxylated fattyacids). The resulting compounds may have altered biological activityand/or bioavailability. Thus, the covalently modified fatty acid can bea pro-drug with reduced biological activity which upon in vivoadministration is slowly transformed into a more active molecule(underivatized LTB₄ agent). Variants may also be metabolically stableand biologically active analogs of LTB₄ agents altered in a way thatwill result in retarded disposition of the compound (decreasedmetabolism and/or elimination). Variants with modifications at the omegaend (such as 20,20,20-trifluoromethyl-LTB₄) show increased resistance toomega-oxidation (a catabolic process of unsaturated fatty acids); othervariants with modification at the omega end at the level of carbons 13to 20 (such as 19-methyl-LTB₄ or 19,19-dimethyl-LTB₄ or 19-fluoro-LTB₄or 19,19-difluoro-LTB₄ or 18,20-difluro-LTB₄ or 20-fluoro-LTB₄) may showincreased resistance to omega-oxidation and variants with modificationsat the carboxylic end, at the level of carbon 1, 2, 3 or 4 (for example,3-thio-LTB₄, 3-hydroxy-LTB₄, 3-methyl-LTB₄ or 3,3-dimethyl-LTB₄ or3-fluoro-LTB₄ or 3,3-difluoro-LTB₄ or 2,3-difluoro-LTB₄, LTB₄methylsulfonylamide, LTB₄ methylamide), may show increased metabolicresistance to beta-oxidation and/or to elimination (such as uptake byprobenecide-sensitive organic acid transporter). Other variants withmodification(s) at carbon 12, such as 12(R)-methyl-LTB₄, may showincreased resistance to reduction of the 11,12 double bond (a metabolicpathway of LTB₄). Other variants are analogs of LTB₄ agents withstructural changes, such as changes in chain length (chain lengthincreased or decreased by up to 4 carbons), addition of double bond(s),saturation of double bond(s), changes in double bond(s) geometry (cis totrans or vice versa), change of double bond(s) for triple bond(s),change in the configuration of one or several functional group(s) (R toS or S to R), or where one or several functional group(s) orsubstituent(s) are either removed, added or changed for other functionalgroups or substituents (including but not limited to hydroperoxyl,carbonyl, sulfhydryl, sulfoxide, sulfone, cysteinyl, glutathionyl,cysteinyl-glycine, methyl, isopropyl, benzyl, chloro, fluoro), or wherethe positions of one or several functional groups and/or one or severaldouble bonds has been moved by one, two or three carbons relative to theomega end. The LTB₄ agent may be a variant carrying one or several ofthe above mentioned structural modifications.

The LTB₄ agents and variants of LTB₄ agents are structurally related toLTB₄ and bind or may bind with different affinities to either the cellsurface binding sites of LTB₄ (or other related eicosanoids, includingbut not limited to 5-HETE, LTD₄, lipoxin A₄) present on variousleukocytes (and other cell types), or to the nuclear binding site ofLTB₄, the transcription factor PPARα (peroxisome proliferator-activatedreceptor alpha) (Devchand P. R., et al., Nature 384:39, 1996), or toother unknown binding sites of LTB₄, resulting in the expression of thebiological activities of LTB₄ and LTB₄ agents. The LTB₄ agents andvariants show or may show biological activities qualitatively similar tothat of LTB₄ (but may be more or less active than LTB₄ itself) and thuscan be expected to exert an adjuvant activity similar to that of LTB₄.The LTB₄ agents and variants thereof are included within the scope ofthis invention.

The term LTB₄ agent also includes agents not structurally related toLTB₄ including but not limited to the chemotactic peptideformyl-met-leu-phe (fMLP) (and analogs such as N-formyl-nle-leu-phe,N-formyl-met-leu-phe-benzylamide, N-formyl-met-leu-phe-methyl-ester andN-formyl-Nle-leu-phe-nle-tyr-lys), the complement fragment C5a andanalogs, and the biologically active phospholipid platelet-activatingfactor, 1-0-hexadecyl-2-0-acetyl-sn-glycero-3-phospho-choline (andanalogs such as 1-0-octadecyl-2-0-sn-glycero-3-phosphocholine and1-0-hexadecyl-2-N-methyl-carbamyl-sn-glycero-3-phosphocholine) thatstimulate or may stimulate the release of unsaturated fatty acids incells (mainly arachidonic acid) and consequently the formation of one orseveral LTB₄ agents, and may therefore exhibit an adjuvant activitysimilar to that of LTB₄. The above-mentioned LTB₄ agents notstructurally related to LTB₄ are thus included within the scope of thisinvention.

The term LTB₄ agent also includes formulations of compounds which mightcontain a mixture of two or several LTB₄ agents or an LTB₄ agent and oneor several equally or less active isomer(s) of the LTB₄ agent(positional, geometrical or optical isomers).

The term LTB₄ agent also includes antibodies to the LTB₄ receptor, oranti-idiotypic antibodies to antibodies raised against LTB₄ or one ofthe above-mentioned analogs or variants of LTB₄, which can be expectedto elicit an LTB₄-like biological response, such as an antiviral effect.

ii) Vaccines

The vaccines for which LTB₄ can be used as an adjuvant include allvaccines available for humans and animals. The expression “vaccine” isintended to include any types of preparations (purified or recombinantproteins, whole-inactivated microorganisms, fragmented microorganisms,live-attenuated microorganisms, complex sugars, etc). The expression“microorganisms” includes DNA and RNA viruses, retroviruses, bacteria,parasites and fungi.

To test the adjuvant potential of LTB₄, groups of 10 BALB/c mice wereimmunized once by intramuscular injection with 5 μl of the commercial“Fluviral” vaccine preparation in combination with saline or 1 ng ofLTB₄. Fourteen days following immunization, mice were bled and seraanalyzed for specific anti-Influenza IgG antibodies by ELISA. Theresults obtained indicate that mice that received LTB₄ concomitantlywith the vaccine developed a greater anti-Influenza antibody response(*p<0.05) in all of the three dilutions tested. A dose of 1 ng of LTB₄was found to be optimal. These results clearly show that a simpleaddition of LTB₄ to the vaccine preparation can positively influencespecific antibody production.

Another vaccine preparation that would greatly benefit from an adjuvantis the BCG vaccine against tuberculosis. This live attenuated vaccinefrom Pasteur-Merieux Connaught is weakly immunogenic requiring multipleadministrations in order to induce anti-BCG antibodies. We testedwhether LTB₄ could influence anti-BCG antibody development. BALB/c mice(n=4-5/group) were immunized intradermally four times with the vaccinepreparation in combination or not with varying doses of LTB₄ (1-10 ng).Mice were immunized on day 0, 24, 68 and 145. On day 160, mice were bledand sera analyzed for anti-BCG antibodies by ELISA. The results indicatethat sera from LTB₄ treated mice had higher levels of anti-BCGantibodies than the BCG+saline group, over all dilutions of sera tested.These results clearly show that a weak vaccine, such as the BCG vaccine,can greatly benefit from the adjuvant properties of LTB₄, making it moreefficient at inducing a specific antibody response.

Our next series of experiments were designed to test the potential ofLTB₄ to modulate antibody response during an acute CMV infection. BALB/cmice were infected by intraperitoneal (i.p.) injection with 10,000 pfuof murine CMV. On day 5 post-infection, mice (n=10) received saline byi.p. injection or LTB₄ (5 μg/kg) (n=9) by i.p. injection 3 times a weekfor 12 weeks. At that time, sera was taken from each mouse and testedfor anti-CMV specific antibodies. The results obtained indicate thatmice receiving LTB₄ had more anti-CMV antibodies than mice from theplacebo-treated group indicating that LTB₄ can positively influencedanti-CMV antibody formation.

Lastly, the sera from each mouse infected with CMV and treated withsaline or LTB₄ (see above) were analyzed for neutralizing antibodies.Briefly, a 1/100 dilution of individual serum was incubated with 175 pfuof murine CMV for 1 hour on ice. Samples (sera-virus) were then added tomouse embryonic fibroblast and incubated at 37° C. for 2 hour.Unadsorbed viruses were removed and cells were overlaid with methylcellulose and incubated for 4 days at 37° C. in a humidified atmospherewith 5% CO₂. At this time cells were fixed, colored with violet crystaland the number of plaques (CMV infected foci) counted. A reduction inplaque number indicates that a serum has neutralizing activity.Uninfected mice had no neutralizing antibodies against CMV, as expected.Two out of 10 mice (20%) treated with a placebo were found to possesssera with CMV neutralizing activity. This is in sharp contrast with 78%of sera of LTB₄ treated-mice, which showed CMV neutralizing activity. Wenext compared the neutralizing activity of sera that tested positive forCMV neutralization. The 2 sera of infected mice that received salinewere able to reduce, in average, CMV infectivity by 24%. In contrast,the 7 sera of LTB₄ treated-mice were found capable of reducing CMVinfectivity by 45%, almost twice the activity of control mice.

The present invention will be more readily understood by referring tothe following examples which are given to illustrate the inventionrather than to limit its scope.

Example I Assay for Anti-Influenza Antibody Generation in BALB/c MiceFollowing Immunization With the “Fluviral” Vaccine

Adult (6-8 weeks) BALB/c female mice were immunized by intramuscularinjection with 5 μl of the commercial Influenza (Fluviral) vaccine incombination or not with 1 ng of LIB₄. Fourteen days later, mice werebled by cardiac puncture and sera obtained. Anti-Influenza antibodieswere quantitated by enzyme-linked immunosorbent assay (ELISA). Wells ofa 96-well plate were coated with the Fluvial vaccine preparation ( 1/100dilution) in 0.1 M carbonate buffer (pH 9.0) by overnight incubation at4° C. Wells were washed with Tris-buffered saline with 0.1% Tween-20(TBS-T) followed by the addition of 100 μl of increasing dilutions ofthe sera to be tested. After a 2-hour incubation at room temperature,the wells were washed six times with TBS-T. One hundred μl ofalkaline-labeled goat anti-mouse IgG were added to each well andincubation allowed to proceed for one hour at room temperature. Wellswere washed six more times with TBS-T followed by the addition of OPDsubstrate and developer solution. After 30 minutes, the absorbance (405nm) from each well was recorded using an ELISA plate reader. The values,expressed as optical density (OD), were plotted against the reciprocalof serum dilution. Results show the mean OD of sera from 5 animals pergroup+S.D. in one experiment representative of two (2) other. As shownin FIG. 1, mice receiving a combination of vaccine and 1 ng of LTB₄generated a significantly higher anti-Influenza antibody response whencompared to the mice receiving the vaccine and saline.

Example II Assay for Anti-Mycobacterium Tuberculosis Antibody Generationin BALB/c Mice Following Immunization With the BCG Vaccine

Adult (6-8 weeks) female BALB/c mice were immunized by intradermicinjections with 25 μl of the anti-tuberculosis BCG vaccine incombination or not with varying doses (1-10 ng) of LTB₄. In all cases,the injected volumes were brought up to 100 μl with saline. Mice wereimmunized on days 0, 24, 68 and 145. On day 160, mice were bled bycardiac punctures and sera tested for anti-mycobacterium tuberculosisantibodies. ELISA plates were coated with 1/50 dilution of the BCGvaccine preparation in 0.1 M carbonate buffer (pH 9.0) by overnightincubation at 4° C. Wells were washed with TBS-T and non-specific siteswere blocked by the addition of saline containing 10% fetal bovine serumfor one hour at room temperature. After several washes, 100 μl ofincreasing dilutions of the sera to be tested were added to each well.As a negative control (background), the serum of a naïve BALB/c mousewas used. After a 2-hour incubation at room temperature, the wells werewashed six times with TBS-T. One hundred μl of alkaline-labeled goatanti-mouse IgG were added to each well and incubation allowed to proceedfor one hour at room temperature. Wells were washed six more times-withTBS-T followed by the addition of OPD substrate and developer solution.After 30 minutes, the absorbance (405 nm) from each well was recordedusing an ELISA plate reader. The values, expressed as optical density(OD), were plotted against the reciprocal of serum dilution. Results(FIG. 2) show the mean OD of each group of serum. In average, sera ofmice receiving a combination of BCG vaccine and LTB₄ had greaterreactivity against mycobacterium antigens than the group receivingBCG+saline, indicating a higher specific antibody response.

Example III Assay for Anti-CMV Antibody Generation Following Acute CMVInfection in BALB/c Mice

Total Anti-CMV Antibodies

Adult (6-8 weeks) female BALB/c mice were infected by intraperitoneal(I.P.) injection with 1×10⁴ pfa of murine CMV. Starting on the fifth daypost-infection, mice were injected with saline or LTB₄ (5 μg/kg) by I.P.injection at a frequency of three times a week for 12 weeks. Mice werethen bled and sacrificed. Total anti-CMV antibodies from each mouse werequantified by ELISA. ELISA plates were coated with a lysates fromCMV-infected fibroblasts as the source of CMV antigens. One μg ofprotein lysates were added to each well and incubated-overnight at 4° C.After several washes with TBS-T, non-specific sites were blocked by theaddition of saline containing 10% fetal bovine serum for one hour atroom temperature. Wells were rinsed 3 times with TBS-T and reacted withdiluted sera preparations from each mouse. Sera were allowed to reactfor 2 hours at room temperature with the serum of a naïve (uninfected)BALB/c mouse used as negative control. After a 2-hour incubation at roomtemperature, the wells were washed six times with TBS-T. One hundred μlof alkaline-labeled goat anti-mouse IgG were added to each well andincubation allowed to proceed for one hour at room temperature. Wellswere washed six more times with TBS-T followed by the addition of OPDsubstrate and developer solution. After 30 minutes, the absorbance (405nm) from each well was recorded using an ELISA plate reader. The values,expressed as optical density (OD), were plotted against the reciprocalof serum dilution. Results (FIG. 3) represent the mean and standarderror derived from 9 data points for each group. The results obtainedindicate that mice receiving LTB₄ had more anti-CMV antibodies than micefrom the saline-treated group indicating that LTB₄ can positivelyinfluenced anti-CMV antibody formation.

Neutralizing CMV Antibodies

The serum from each mouse, described above, were tested for their CMVneutralizing activity. Briefly, a 1/100 dilution of individual serum wasincubated with 175 pfu of murine CMV for 1 hour on ice. Samples(sera-virus) were then added to mouse embryonic fibroblast and incubatedat 37° C. for 2 hour. Unadsorbed viruses were removed and cells wereoverlaid with methyl cellulose and incubated for 4 days at 37° C. in ahumidified atmosphere with 5% CO₂. At this time cells were fixed,colored with violet crystal and the number of plaques (CMV infectedfoci) counted. A reduction in plaque number indicates that a serum hasneutralizing activity. Uninfected mice had no neutralizing antibodiesagainst CMV, as expected (Table 1). Table 1 illustrates the effects ofprolonged LTB₄ administration on neutralizing anti-CMV antibodygeneration during acute CMV infection in BALB/c mice.

TABLE 1 Effects of LTB₄ administration of the generation of CMVneutralizing antibodies Mice sera with CMV-neutralizing activity (%) Na

ve 0/10 (0%) (uninfected) Placebo 2/10 (20%) LTB₄ 7/9 (78%)

Two out of 10 mice (20%) treated with saline were found to possess serawith CMV neutralizing activity at a 1/100 dilution. This is in sharpcontrast with 78% ( 7/9) of sera from LTB₄ treated-mice, which showedCMV neutralizing activity (Table 1). We next compared the neutralizingactivity of sera that tested positive for CMV neutralization. The 2 seraof saline treated-mice were able to reduce, in average, CMV infectivityby 24%. In contrast, the 7 sera of LTB₄ treated-mice were found capableof reducing CMV infectivity by 45%, almost twice the activity of controlmice.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth, and as follows in the scopeof the appended claims.

1. A vaccine preparation for enhancing immune response in an individual,which comprises an immune-enhancing effective amount of an leukotrieneB4 (LTB₄) agent in association with a vaccine, wherein said LTB₄ agentis LTB₄ or a salt thereof.
 2. The vaccine preparation of claim 1,wherein said vaccine is immunoprotective against a pathogen selectedfrom the group consisting of Influenza and Tuberculosis.
 3. The vaccinepreparation of claim 1 wherein the salts are the sodium or potassiumsalts of the LTB₄ agent.
 4. The vaccine preparation of claim 2 whereinsalts are the sodium or potassium salts of the LTB₄ agent.